DAT is an integral membrane protein found at the synapse of dopaminergic neurons, and is the primary mechanism of dopamine clearance. This physiologic role of DAT has profound clinical implications in such areas as drug abuse, Parkinson's disease, and schizophrenia. The ability of DAT to transport dopamine is blocked by cocaine, amphetamine and methphenidates. The research proposal focuses on understanding the molecular interactions involved with inhibitor and substrate binding and transport. Specific Aim One examines conformational changes associated during ligand binding and substrate transport through limited proteolysis and epitope-specific immunoblotting. Specific Aim Two characterizes the ability of partially proteolyzed DAT to transport [3H] dopamine and bind [3H] CFT. Specific Aim Three will involve probing the transmembrane domain arrangement within DAT using chemical cross-linkftaers. This research proposal, using of a variety of techniques, is intended to advance the knowledge of DAT structure and function relationships.